Aligned and electrospun piezoelectric polymer fiber assembly and scaffold

ABSTRACT

A method of manufacturing and/or using a scaffold assembly for stem cell culture and tissue engineering applications is disclosed. The scaffold at least partially mimics a native biological environment by providing biochemical, topographical, mechanical and electrical cues by using an electroactive material. The assembly includes at least one layer of substantially aligned, electrospun polymer fiber having an operative connection for individual voltage application. A method of cell tissue engineering and/or stem cell differentiation that uses the assembly seeded with a sample of cells suspended in cell culture media, incubates and applies voltage to one or more layers, and thus produces cells and/or a tissue construct. In another aspect, the invention provides a method of manufacturing the assembly including the steps of providing a first pre-electroded substrate surface; electrospinning a first substantially aligned polymer fiber layer onto the first surface; providing a second pre-electroded substrate surface; electrospinning a second substantially aligned polymer fiber layer onto the second surface; and, retaining together the layered surfaces with a clamp and/or an adhesive compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 12/969,076, filed Dec. 15, 2010, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 61/286,484, filedDec. 15, 2009. The contents of the foregoing applications are herebyincorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made in part by employees of the United StatesGovernment and may be manufactured and used by or for the Government ofthe United States of America for governmental purposes without thepayment of any royalties thereon or there for.

BACKGROUND OF THE INVENTION

Current scaffold designs and materials do not provide all of theappropriate cues necessary to mimic in vivo conditions for tissueengineering and stem cell engineering applications. It has beenhypothesized that many biomaterials, such as bone, muscle, brain andheart tissue exhibit piezoelectric and ferroelectric properties. Typicalcell seeding environments incorporate biochemical cues and more recentlymechanical stimuli. However, electrical cues have just recently beenincorporated in standard in vitro examinations. In order to developtheir potential further, novel scaffolds are required to provideadequate cues in the in vitro environment to direct stem cells todifferentiate down controlled pathways or develop novel tissueconstructs. A scaffold that provides electrical stimuli in conjunctionwith biochemical and mechanical cues will have a significant impact onthe proliferation and differentiation of stem cells and tissueconstructs that can be engineered.

BRIEF SUMMARY OF THE INVENTION

In view of the foregoing, it is an object of the invention to provide ascaffold assembly and related methods of manufacturing and/or using thescaffold for stem cell culture and tissue engineering applications inorder to at least partially mimic a native biological environment byproviding biochemical, topographical, mechanical and electrical cues byusing an electroactive material.

It is a related object of the invention to provide the ability fordelivering electrical and mechanical stimuli through bioactive fibersfor stem cell tissue engineering. Potential applications include stemcell therapy treatment methods include, for example, spinal corddisorders, autoimmune diseases, and Parkinson's disease. Potentialapplications also include, for example, tissue engineering constructsfor myocardial infarcts, blood vessels, and skin grafts.

These objects are achieved by the present invention, which in oneembodiment provides an assembly for tissue engineering and/or stem celldifferentiation using electrical and/or mechanical stimuli throughbioactive fibers comprising at least one layer of substantially aligned,electrospun polymer fiber having an operative connection for individualvoltage application.

In another embodiment, the invention provides a method of cell tissueengineering and/or stem cell differentiation, said method including thesteps of providing an assembly having at least two layers ofsubstantially aligned, electrospun polymer fiber having an operativeconnection for individual voltage application to each layer; seeding theassembly with a sample of cells suspended in cell culture media;incubating for an effective time period; applying an effective voltageto one or more layers; and recovering cells and/or a tissue construct.

In yet another embodiment, the invention provides a method ofmanufacturing an assembly for tissue engineering and/or stem celldifferentiation including the steps of providing a first pre-electrodedsubstrate surface; electrospinning a first substantially aligned polymerfiber layer onto the first surface; providing a second pre-electrodedsubstrate surface; electrospinning a second substantially alignedpolymer fiber layer onto the second surface; and, retaining together thelayered surfaces with a clamp and/or an adhesive compound.

Additional objects, embodiments and details of this invention can beobtained from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 illustrates SEM micrographs of electrospun PVDF after (a)non-woven image collected on a static plate without auxiliary electrodeuse, (b) 1-2 seconds, (c) 5 minutes, (d) 15 minutes, (e) 30 minutes, (f)45 minutes [magnification 500× (a,c,d,f), 100× (b), and 845× (e)].

FIG. 2 illustrates scaffolds fabricated from CP2 polyimide with (a)optical image of 4 layer aligned fibers [mag. 10×], (b) live/dead assayresults on aligned fibers, (c) optical image of nonwoven fibers [mag.10×], and (d) live/dead assay results on nonwoven fibers.

FIG. 3 illustrates SEM micrograph of a cell attached to the surface of aCP2 fiber.

FIG. 4 illustrates a schematic drawing of an exemplary scaffold withPVDF fibers electrospun directly onto a pre-electroded surface.

FIG. 5 illustrates a schematic drawing of an assembly of four scaffoldlayers in a 0/90/+45/−45 configuration.

DETAILED DESCRIPTION OF THE INVENTION

Stem cells have enormous therapeutic potential for treating a multitudeof medical disorders such as Parkinson's disease, autoimmune diseases,and spinal cord injuries. An attractive feature of this therapy is theability to inject the stem cells directly at the treatment locationwithout the need for additional delivery mechanisms. Adult stem cellsare currently used to treat leukemia and other blood and bone disordersand recently have been approved as a treatment strategy for myocardialinfarcts and degenerative joint disease. Human mesenchymal stem cells(hMSCs) are adult stem cells derived from bone marrow that havedemonstrated remarkable multipotency through their ability todifferentiate across germ layers. A great deal of research in this areahas focused on their trans-differentiation potential and the ability todirect their differentiation to specific lineages. A key componentinfluencing the differentiation fate of stem cells is the in vitroenvironment in which they are cultured for expansion. This environmentis comprised of a multitude of factors with the fundamentals being theselection of media, 2-D vs. 3-D scaffolds, and material considerations.Several groups have identified key differences in employing mediacontaining serum and that free of serum with varying results. Thephysical culture conditions, however, remain rather elusive withnumerous variables to consider such as topography, spatial dimensions,material chemistry and mechanical properties. Current scaffold designsand materials do not provide all of the appropriate cues necessary tomimic in vivo conditions. It has been hypothesized that manybiomaterials, such as bone, muscle, brain and heart tissue exhibitpiezoelectric and ferroelectric properties. Typical cell seedingenvironments incorporate biochemical cues and more recently mechanicalstimuli, however, electrical cues have just recently been incorporatedin standard in vitro examinations. In order to develop their potentialfurther, novel scaffolds are required to provide adequate cues in the invitro environment to direct the stem cells to differentiate downcontrolled pathways. A scaffold that provides electrical stimuli inconjunction with biochemical and mechanical cues will have a significantimpact on the proliferation and differentiation of the stem cells.

The primary objectives of this invention are twofold; first, to developa novel scaffold that provides mechanical and electrical cues that moreclosely mimic the cells' native environment (such as heart, brain,nerve, muscle) and second, to determine the influence of the scaffold onthe differentiation potential of exemplary human mesenchymal stem cells.The inventive embodiments are segmented into four key parts; the firstis the manufacture of aligned electroactive fibers that will provide theelectrical and mechanical cues; the second is to create a 3-D structurefrom the aligned electroactive fibers and to design a scaffold thatcombines the 3-D aligned fiber architecture with electrical andmechanical stimulus capability; the third part is to determine theeffect of topography on exemplary human mesenchymal stem cellsphenotypic development by comparing 2-D vs. 3-D environments; and thefourth is to determine hMSC phenotypic development as a function ofelectrical and mechanical stimuli application.

In at least one embodiment, the invention involves the fabrication ofelectroactive polymer fibers. Electrospinning was used to yield fibersthat can exhibit crystalline structures in polar form due to the strongelectric field. And since aligned fibers are crucial in directing stemcell differentiation, the electrospinning process was modified togenerate highly aligned electroactive fibers in situ using an auxiliaryelectrode to focus the electric field. For more details on an exemplarymethod and system for aligning fibers during electrospinning, please seeapplication Ser. No. 12/131,420, which has published as US 2009/0108503A1, which is herein incorporated by reference.

One of the specific aims of this invention is to fabricate an exemplaryscaffold that confirms the effect of topography and architecture on thedifferentiation of exemplary hMSCs. Although hMSCs are discussed indetail, certain inventive embodiments include other types of stem cells,e.g. induced pluripotent and/or embryonic stem cells. Research performedby Yim and Hu et. al. demonstrated the significant influencetopographical cues have in directing the differentiation of hMSCs. Itwas observed that hMSCs respond to features in the nanometer andmicrometer range. The formation of the extracellular matrix has beenshown to occur on the nanometer level making this an attractivedimension. Other groups have investigated the effect of surfacechemistry on the attachment, proliferation and differentiation of hMSCs.Engler et. al. reported that a material's elastic modulus alone iscapable of directing stem cell differentiation. They found that collagenproduction was significantly reduced when hMSCs were cultured on a‘soft’ matrix compared to a ‘hard’ matrix intended to mimic thedifferences between biomaterials such as the brain and bone. In additionto topography, the architecture contributes considerably to the overallsuccess of the scaffold. A scaffold comprising a three dimensionalenvironment more closely mimics native surroundings than one constructedfrom two dimensions. There are two predominant scaffold constructs thatprovide a three dimensional environment, gels and fibers. Gels producedfrom collagen, fibrin, gelatin, alginate, and more recentlythermoresponsive polymers demonstrate differing results compared toconventional two dimensional systems when used to culture hMSCs.Scaffolds fabricated from fibers with diameters ranging from nanometersto micrometers have also been investigated extensively as threedimensional constructs. Interesting results have been observed with thistype of environment. One study compared the effects of a threedimensional gel environment to a fibrous scaffold and found vastlydiffering results between the two. Another scaffold was designed tomimic native cartilage by employing a composite structure manufacturedto emulate the mechanical properties. A key issue confronting threedimensional scaffolds to date is control over the porosity. Porosity isvital to the health and maintenance of the cells in culture. If theporosity is insufficient, the cells will not receive adequate nutrientsand may undergo hypoxia. One way to prevent this is to use alignedfibers to control porosity. Aligned fibers have been investigatedextensively and shown to promote cell alignment and attachment due totheir high surface to volume ratio. Studies suggest that a fibrousstructure plays a fundamental role in the modeling of the extracellularmatrix and overall gene expression.

Some embodiments of the invention involve electroactive polymer fibers,and in preferred embodiments, the polymer fibers used are composed ofpolyvinylidene fluoride (PVDF). PVDF is a commercially available polymerused in a variety of areas ranging from aerospace, medical, andautomotive to common household applications. It is a crystallinematerial capable of assuming four different phases (α, β, γ, δ)depending upon processing and post-processing conditions. The γ and δphases are quite uncommon and will not be examined for this study. Themost common form is the α-phase. The crystalline structure is in atrans-gauche (TGTG) configuration. When the material is mechanicallystretched its crystalline form is altered to assume an all-transconfiguration, which renders it electroactive due to alignment of thedipoles present in the structure. Subjecting the mechanically stretchedmaterial to an electric field further increases the dipole alignment inthe crystalline structure and enhances the electroactive properties.This highly polar form is classified as the l-phase and is the desiredstate due to its unique electroactive properties. Electroactive PVDF isin a class of materials that exhibit piezoelectricity, i.e. a mechanicalstrain is elicited with the application of a voltage and conversely, anelectrical signal is produced with the application of a mechanicalstrain. It is also pyroelectric, exhibiting an electric charge as afunction of temperature. This is typically referred to as ferroelectric.PVDF poses an exciting possibility for cell culture studies for at leasttwo reasons. First, the piezoelectric properties of the β-phase allowsfor direct application of electrical and mechanical stimuli to thecells. Second, its pyroelectric property resulting from an appliedtemperature is novel. Thermally stimulated current (TSC) data indicatesthat PVDF generates a slight current equivalent to approximately 2.2E-10A/m2 when subjected to standard cell culture conditions of 37° C. Thisproperty was included in the analysis during the topographical portionof the study. PVDF has been considered for a wide range of biomedicalapplications such as sutures and surgical meshes due to its inertchemistry and good biocompatibility.

Exemplary human mesenchymal stem cells (hMSCs) have generated anenormous amount of interest due to their multipotency, the fact thatthey are non-controversial and they do not form teratomas. hMSCs havedemonstrated multipotent potential through their ability totrans-differentiate. Several research groups have reported success indirecting hMSCs to differentiate into adipocytes, chondrocytes,osteoblasts, neurons, cardiomyocytes and muscle. There is considerabledebate as to whether hMSCs actually trans-differentiate or are coercedinto specific lineages by fusion with mature cells present in theirsurroundings. Research by Engler et.al. has provided additionalclarification surrounding this highly controversial topic with theirdiscovery on the multipotent potential of hMSCs based on the modulus ofthe scaffold. Their study revealed a passive response of the hMSCs tothe microenvironment which is presumed to be indicative of a multipotentstem cell.

Although hMSCs trans-differentiation potential has sparked a great dealof debate among the research community, there is no dispute regardingthe multipotent potential of hMSCs and their ability to be consideredfor therapeutic applications. In fact, hMSCs are an ideal research linebecause they are not controversial since they are derived from bonemarrow and in many instances, they can be autologous, eliminatingimmunorejection concerns. Embryonic stem cells, while undoubtedlypluripotent, have generated a significant degree of controversyprimarily over sourcing. It will be challenging for human embryonic stemcells to be considered for clinical applications in the near term forthis reason and also because they are extremely difficult to control,often giving rise to tumor formation.

In order to direct stem cell differentiation in vitro, it is necessaryto provide the appropriate cellular cues and environment. This includesbiochemical, mechanical and electrical signals that emulate the nativecellular environment. Typically, cells are seeded in a tissue culturetreated polystyrene dish and provided with biochemical cues to guidethem down a specific lineage. This has demonstrated moderate success,but does not emulate the cells native environment. Researchers haverecently begun to investigate the effects of mechanical properties onthe growing cells. They have demonstrated that providing a mechanicalenvironment similar to a cell's native area contributes significantly tothe lineage pathway chosen. Another group of researchers has shown thatproviding electrical signals to embryonic stem cells results in a muchlarger number of cells exhibiting markers for a neuronal pathway. Inorder for stem cell therapy to become viable, cells must be harvested,dissociated into individual cells and expanded ex vivo. Stem cells thatcan be differentiated into a preferred lineage and expanded down thatpathway possess the ability to provide great therapeutic potential fornumerous health disorders. hMSCs are currently being considered fortreatment in Parkinson's disease and other neural disorders due to theirdemonstrated ability to trans-differentiate or by creating a favorableenvironment through the release of soluble factors.

There are a multitude of neurological and immune disorders that, despitesociety's best efforts, their cures remain elusive in the researchcommunity. In order to treat these conditions, new methods and clinicaltreatments must be considered. Technology has brought about substantialmedical advances through the introduction of state-of-the-art diagnosticequipment and the ever changing drug therapies available. It isnecessary to step around some of the current barriers to treatment andexamine new options. Stem cell therapy offers boundless potential forimproving the quality of life for millions of individuals, possibly evenoffering cures for diseases previously unattainable. Hence, despitenumerous obstacles, research into hMSCs for stem cell therapy remainsrobust.

Inventive materials selected included PVDF, which was selected for itsunique electroactive properties and its potential for biocompatibility.A fluorinated polyimide, CP2, was also selected and synthesized from2,2′-Bis(3,4-dicarboyxphenyl)hexafluoropropane dianhydride (6FDA) and1,3-bis(4-aminophenoxy)benzene (APB) and was included to compare to thePVDF due to its potential for biomedical applications. Thebiocompatibility of electroactive PVDF was verified by performing alive/dead assay and examining the metabolic activity compared tostandard tissue cultured polystyrene (TCPS) and a polyimide, CP2. Thecells demonstrated good spreading and morphology on both film surfaces.A WST-1 assay confirmed the metabolic activities of PVDF and CP2 werecomparable to TCPS indicating good biocompatibility between hMSCs andPVDF. Since the surface of the material plays a critical role in thecell attachment and spreading, we analyzed the films for functionalgroups present, surface roughness, and surface energy. FTIR-ATR resultsindicated the presence of primary aliphatic —OH functional groups forPVDF by the peaks present at 1066 cm-1 and ˜3600 cm-1. Aliphatic —COOHfunctional groups were identified in the spectra for CP2 by the broad—OH stretching region from 2500-3500 cm-1 and peaks present at 1072cm-1, 1239 cm-1 and 1720 cm-1. It has been reported that —OH and —COOHfunctional groups play a significant role in cell attachment andproliferation. Several research groups have attempted to add thesefunctionalities to their materials to enhance these features and promoteproliferation.

Contact angle measurements were performed on each film surface usingmedia warmed to 37° C. and found to be 67.34° for PVDF and 78.68° forCP2 at equilibrium indicating the surfaces were hydrophilic. The surfaceenergy was calculated based on the equilibrium contact angle observedand was found to be 34.91 dynes/cm for PVDF and 26.02 dynes/cm for CP2.These values are quite low for a hydrophilic material however, thesurface roughness is not included in the theoretical equation althoughit contributes significantly to the wettability of the surface. Theaverage surface roughness for each material was found to be 0.069 μm forPVDF and 0.009 μm for CP2 indicating sufficient roughness to promoteadhesion at the cellular level. The results were illustrated with imagesobtained using an optical profilometer.

Several researchers have reported a significant reduction inelectroactive properties over a period of time when exposed to in vivoconditions. Therefore, an in vitro degradation study was performed onPVDF to ensure the material properties were not changed as a result oflong term exposure to the environment.

The mechanical properties were measured on five PVDF samples andaveraged over the degradation period. There was very little change overthe 28 day period with overall about 4% difference between the baselineand the last day for the ultimate break stress, 7% difference in theelongation and about a 5% difference in the modulus. There was not areportable difference in the piezoelectric properties over the 28 dayperiod as indicated by the thermally stimulated current method.

The electrospinning manufacturing process is a simple and versatileprocess that can be used to fabricate micro and nanofibers from polymersolutions and melts. The process has typically produced random nonwovenmats and was modified for this study to develop aligned fibers for amore controlled architecture. The set-up incorporates an auxiliaryelectrode that creates a dipole field and directs the electrospun fiberto a collector without the typical whipping and bending instabilitiesobserved in other systems. The fiber continues to be pulled along thedipole field over time and can be directed at an angle due to therepositioning of the auxiliary electrode. SEM micrographs illustrated inFIG. 1 demonstrate the degree of alignment achieved for various timeperiods ranging from 1 second to 45 minutes.

The crystalline structure of PVDF aids in determining the overallproperties of the resulting polymer, specifically, the piezoelectricstate. A common technique to induce the 3-phase is to subject the αstructure to mechanical stretching and a high electric field whichresults in orientation of the dipoles within the crystalline structure.Electrospinning incorporates both of these features; first bymechanically drawing the fiber from the spinneret to the collector andsecond by creating a strong electric field which the fiber is expelledthrough. The results from electrospinning a pure α-phase powder from asolution of 50/50 DMF:Acetone indicate a transition to the β-phaseoccurred based on peak shifts illustrated in the x-ray diffraction (XRD)diffractogram. When examining the material using XRD, there are twoprimary peaks indicating the starting crystalline form is in the alphaphase. A shift of 20 to 20.6° indicates 200/110 reflections of theβ-phase whereas the two shoulder peaks at 2θ=18.25° and 19.8° representthe 020 reflection of the α-phase. The peak around 2θ=26.63° isrepresentative of the α-phase. This peak was not present in any of theprocessed forms present in the diffractogram indicating a shift from theα-phase to the β-phase occurring. The electrospun nonwoven PVDF showed apeak shift around 2θ=28.57° suggesting it has not fully transitioned tothe β-phase. An additional peak around 2θ=36.14° represents the 200plane and is another indicator that the β structure has been formed.

Fourier transform infrared spectroscopy (FTIR) with attenuated totalreflectance confirmed the transition to the β-phase by a change in thevibrational bands characteristic of the α-phase at 615 cm-1, 766 cm-1and 795 cm-1 and the presence of a vibrational band at 840 cm-1.Differential scanning calorimetry (DSC) results for the electrospunaligned fiber depicted a melting point of approximately 160° C. on thefirst heat. Subsequent quenching and a second heat indicated a shoulderpeak present at 155° C. and 160° C. which was indicative of the twocrystalline phases present, further demonstrating the crystallineβ-phase transformation. The melting temperature for the electrospunaligned fibers was lower than that for the poled film (165° C.). Thereare several factors that may contribute to the slight decrease. Thenumber of head-head and tail-tail chain configurations will play a rolein the overall melting point as will the percent crystallinity and theamount of α-phase and β-phase present. The presence of a shoulder peakin the poled film and its absence in the pure powder further signifiesthe presence of both phases.

A modified Rheovibron was used to measure the d31 piezoelectric constantof a fibrous mat electrospun from PVDF for 45 minutes. Gold electrodeswere deposited on both sides of the mat and measurements were performedby applying a tensile load of 35 g and measuring the d31 constant as afunction of frequency and temperature. Results were obtained forfrequencies at 1 Hz, 10 Hz, 20 Hz and 100 Hz over a temperature range of23° C. to 50° C. A value for d31 was obtained, thus validating therationale that electrospun aligned PVDF fibers are poled in-situ duringthe electrospinning process.

As discussed earlier, topography plays a significant role in cellattachment, differentiation and proliferation of exemplary hMSCs. Inorder to determine the effect of fiber morphology on the culture ofhMSCs, the inventors performed cell culture studies on films of the samematerial to be examined in fiber form. Preliminary results fromexamining the cells after 7 days and 14 days in culture indicateadvanced cell structure by the presence of intermediate filamentvimentin. A strong presence of beta III tubulin (TUJ1), an early stageneuronal marker, was present for all of the materials and MAP2 waspresent in the group conditioned with retinoic acid and PVDF. Retinoicacid has been identified as a chemical agent that induces the neuronallineage in culture. Reverse transcriptase PCR was performed on mRNAextracted from the samples after 7 days and 14 days in culture. Gelelectrophoresis staining indicated the upregulation of October 4, amarker typically present in embryonic stem cells, for the CP2 polyimidecoverslip, coverslip with retinoic acid and TCPS after 14 days inculture. Genes corresponding to C-kit were present in PVDF andcoverslip. All of the material samples expressed SOX 9, an early markerfor chondrycyte formation and alpha-fetoprotein (AFP). AFP is a proteinexpressed during early endoderm development and has been shown toexpress in hMSCs during hepatocyte differentiation. PVDF also indicatedthe upregulation of MAP2 after 14 days in culture. This indicates theremay be something happening on the cell signaling level with PVDF,perhaps related to the pyroelectric behavior, since MAP2 is a geneexpressed during neuronal development. The expression of both endodermand mesoderm markers suggested a mixed population of cells present. Gelelectrophoresis data for the 7 day films did not readily express any ofthe genes the inventors probed for although the housekeeping genes, betaactin and GADPH, were clearly observed.

In order to determine the effect of PVDF fibers on the culture of hMSCs,scaffolds were manufactured from both CP2 polyimide and PVDF. The fiberswere electrospun directly onto rings to be used in culture and affixedusing cyanoacrylate. The fibers were deposited in a configuration toallow the greatest degree of porosity while maximizing cell attachment.Three different aligned fiber configurations were examined. The‘standard’ configuration was composed of fibers having average diametersof approximately 8 μm, the ‘fine’ sample fiber diameters were on theorder of 1 μm and the ‘mixed’ sample consisted of a basement layer offine fibers followed by three additional layers of standard sizedfibers. The lay-up was in a 0/90/+45/−45 arrangement for each sampletype. A sample comprised of a nonwoven collection of fibers was used toassess the impact of aligning the fibers. Initial studies examined theeffect of this architecture by performing live/dead assays on fiberscaffolds manufactured from CP2.

The results are illustrated in FIG. 2. It is apparent from the results,which indicated the presence of some dead cells, that the nonwovenscaffold either did not allow the appropriate nutrients to diffusethrough the scaffold or that the cells could not penetrate the scaffoldfor proper attachment. The aligned fibers, although quiteauto-fluorescent, provide a good environment for cell culture as can beobserved by the cell attachment along the length of the fibers andthroughout the multiple layers. An SEM micrograph depicting a dehydratedcell attached to a CP2 fiber is illustrated in FIG. 3. Gelelectrophoresis results indicated the presence of Sox 9, a marker forchondrocyte formation, for each of the CP2 fiber sizes and architecturesexamined after 7 days in culture. Immunostaining of CP2 aligned fibersfor vimentin, actin and the nucleus revealed a well organized cell. Thecells showed substantial alignment along the fibers. The mixed fibersdid not display a cell structure as organized as those observed on thestandard and fine fibers. The nucleus was much more elongated on thefine fibers compared to the standard fibers. This suggests the cell wassitting on top of the standard size fibers since they are roughly thesame size and the cell was elongating and perhaps wrapping around thefine diameter fibers in order to attach.

PVDF fibers were also electrospun onto rings, in the configurationdescribed above for CP2, and affixed with cyanoacrylate, cultured for 7days and stained for vimentin, actin and the nucleus. The cells wereobserved with confocal microscopy to attach to the fibers. The nucleuswas elongated along the length of the fiber illustrating the influenceof topography on the cell morphology. It was also observed that whatappears to be two cells attached to different fibers bridged across thefibers to contact one another.

In at least one embodiment of the invention, an assembly for tissueengineering and/or stem cell differentiation is provided using a 3-Dscaffold to apply electrical stimuli. In one embodiment, at leastseveral factors were considered in the design of the 3-D scaffold inorder to apply electrical and mechanical stimulation. These included thegeometry, substrate, electroding, adhesive, clamp fixture and fiberdeposition. An exemplary 3-D scaffold was designed such that PVDF fiberscould be electrospun directly onto a pre-electroded (FIG. 4) surfaceminimizing the potential for shorting by electroding the fiberspost-processing. The plan in this instance was to manufacture fourlayers independently, place them in a 0/90/+45/−45 configuration (FIG.5) and retain them with a specially designed clamp. This allowed for thedirect stimulation of each layer independently. The four layer assemblymimicked the architecture of the previous work with the fibersmanufactured onto rings in order to allow for a more direct comparison.Cell culture studies were performed on the scaffolds with theapplication of voltage to each layer.

Accordingly, each substrate layer was fabricated using Kapton™ polyimide(DuPont) by laser cutting a 28 μm sheet of film. Gold electrodes wereevaporated on each substrate. Gold was chosen as the electrode materialdue to its inert properties and excellent biocompatibility. Devcon™silicon adhesive was used to glue the electrospun fibers to theelectroded substrates. Each layer was fabricated separately, glued andallowed to air dry for a minimum of 48 hours. The four separate layerswere joined together using the silicon adhesive and allowed to dry for aminimum of 48 hours prior to cell culture.

A clamp was designed to hold the four layer scaffold construct togetherand keep it suspended from the bottom surface and allow for each layerto be stimulated independently or simultaneously. A grooved portionprovided space for the flexible leads to extend outside of the culturearea and the top piece secured the scaffold together by fitting snuglyinto the grooved piece. The clamp was fabricated from acrylic polymerresin using the stereolithography rapid protyping process.

In order to maintain the existing architecture for comparison purposes,a similar design will be employed with one end of the substrate fixedand the free end mechanically extended in order to strain the PVDFfibers. The substrate will be cut on each side in order to obtain directstraining of the fibers without the influence of the substrate.Mechanically straining the fibers will elicit an electrical signal thatis expected to have a significant impact on the differentiation andproliferation of the hMSCs over time.

Exemplary human mesenchymal stem cells were obtained from TulaneUniversity. A single male donor was used throughout the study to preventdonor-donor variables. hMSCs were cultured in alpha minimum essentialmedium (αMEM) with L-glutamine, but without ribonucleosides ordeoxyribonucleosides (Invitrogen/GIBCO), containing 16.5% fetal bovineserum, premium select, hybridoma qualified, not heat inactivated (FBS,Atlanta Biologicals), 200 mM in 0.85% NaCl of L-glutamine(Invitrogen/GIBCO), 100 units/ml penicillin and 100 μg/ml streptomycin(Invitrogen/GIBCO). Cell culture medium was filtered through a sterilefilter unit. Cells were plated at an initial density of approximately350 cells/cm² in a 15 cm dish and incubated at 37° C. with humidified 5%CO₂. Media was replenished every three days. Cells were passaged afterreaching approximately 80% confluence by aspirating media, washing 1×with phosphate buffered solution (PBS, Invitrogen) and lifting with0.25% Trypsin and 1 mM ethylene diamine tetracetic acid (EDTA) in Hank'sbalanced salt solution (Invitrogen/GIBCO) for 1-3 minutes at roomtemperature. After incubation in Trypsin/EDTA, 5 ml of cell culturemedia was added and the mixture was transferred to a 15 ml conicalcentrifuge tube. The cells were centrifuged for 10 minutes at 450×g atroom temperature. The supernatant was aspirated and the cell pelletresuspended in 1.0 ml cell culture media. Cells were counted by adding10 μl 0.4% Trypan Blue solution to 10 μl of cell suspension anddispersing them onto a hemocytometer. Cells were seeded onto scaffoldsbetween passages 2-5 at a density of approximately 125,000 cells/cm².Attachment of the cells to the scaffold was promoted by seeding aninitial volume of 250 μl of cell suspension on each scaffold andallowing it to incubate at 37° C. with humidified 5% CO₂ for a minimumof one hour. After the initial incubation period, an additional volumeof 3.4 ml of cell culture media was added to each well and the scaffoldwas placed back in the incubator at 37° C. with humidified 5% CO₂ for 7days. Media was replenished every three days. Electrical leads clampedto the flexible leads of the scaffold were attached to a power supplyand a 9 mV/cm stimulus was applied to each scaffold layer afterapproximately 24 hours post-seeding at a frequency of 500 mHz.

In order to determine an exemplary minimum voltage required to cause aneffect on the calcium channels, a preliminary calcium imaging experimentwas performed using a Fluo-4 NW (no wash) assay (Molecular Probes). PVDFand CP2 polyimide films were examined. Films were laser cut to provide aflexible lead and Au electrodes were evaporated on the surface in orderto attach wires to the substrates. hMSCs were seeded on PVDF and CP2films at a density of 5000 cells/cm² and allowed to incubate for 24hours at 37° C. in humidified 5% CO2. A 250 mM stock solution ofprobenicid was prepared by adding 1 mL of assay buffer to probenicid andvortexing until dissolved. The dye loading solution was prepared byadding 10 mL assay buffer and 100 μL probenicid stock solution toComponent A and vortexing for 1-2 minutes. A 1 M acetylcholine (AcH)(Sigma-Aldrich) sample was prepared in buffer. To prepare for imaging,media was removed from the wells and the samples were washed 2× withPBS. A volume of 1 mL of dye loading solution was added to each sampleand the samples were incubated at 37° C. in humidified 5% CO2 for 30minutes. The samples were placed under an upright fluorescent microscope(Zeiss Axio Observer) fitted with a heated stage. Wires were attached tothe flexible electrodes protruding outside of the chamber. The voltagesupply (Keithley) was monitored using an oscilloscope (Wavetek). Datawas recorded for a period of 2 minutes prior to the application ofstimuli. AcH was added after several minutes of baseline data collectionand served as a positive control. Power was applied to the substrate andrecorded for several minutes. Data was obtained and post-processed usingMetamorph software (Zeiss). The average fluorescence intensity for aminimum of 7 cells was plotted as a function of time.

Results from the live cell calcium imaging study indicated thatapplication of 9 mV/cm of a direct current electric field was sufficientto elicit a response in the cells. The addition of AcH showed a peakintensity of about 600 while the CP2 film with 9 mV/cm direct currentelectric field applied had an intensity of around 1100, nearly two timesthe results obtained with AcH. The PVDF film peaked around 625, similarto the results obtained using AcH to stimulate the Ca+ channels.Spontaneous Ca+ signals were observed in the PVDF film prior to theapplication of 9 mV/cm direct current electric field.

The proliferation of cells was analyzed using EdU Click-iT™ Imaging Kit(Invitrogen). Cell culture was performed as described above. Briefly,hMSCs were seeded on scaffolds and in a 6 well tissue culture dish at adensity of 200,000 cells. After 24 hours in culture, EdU was added tothe media at a concentration of 10 μM. After 3 days in culture, mediawas replenished with fresh media containing 10 μM of EdU. After 7 days,the samples were removed, fixed with 4% PFA (Sigma-Aldrich) for 15minutes at room temperature then washed 2× with PBS containing 3% bovineserum albumin (BSA, Sigma-Aldrich). After the wash was removed, thesamples were incubated for 20 minutes at room temperature in 0.5%TritonX-100 in PBS. The reaction cocktail was prepared by mixing 1.8 mL1× Click-IT reaction buffer, 80 μL CuSO₄, 5 μL Alexa Fluor azide, and200 μL reaction buffer additive. The permeabilization buffer was removedand the samples were washed 2× with PBS containing 3% BSA. The wash wasremoved and the Click-iT reaction cocktail was added to each sample. Thesamples were incubated for 30 minutes at room temperature protected fromlight. The reaction cocktail was removed and the samples were washedwith PBS containing 3% BSA. Each sample was washed with PBS prior to DNAstaining. A Hoechst 33342 (1:2000) was added to each sample andincubated for 30 minutes at room temperature protected from light. Thesamples were washed 2× in PBS and mounted to coverslip with gel mount(Invitrogen). The samples were imaged using confocal microscopy. Aminimum of 400 cells were counted.

Proliferation results for the PVDF fiber stimulated scaffold and TCPSindicated the stimulated scaffold had 10% greater incorporation of EdUcompared to TCPS, with 33% EdU-positive cells in the stimulated fiberscaffold compared to 23% on TCPS.

Immunostaining was performed. After 7 days in culture, samples werewashed with PBS and fixed in 4% paraformaldehyde for 15 minutes at roomtemperature. Following fixation, the samples were washed 2× in PBS. Asolution containing 0.25% Triton-X 100, 1% bovine serum albumin and 10%goat serum was prepared in PBS. Samples were probed for primaryantibodies including Vimentin (1:200, Sigma-Aldrich), F-Actin (1:50,Molecular Probes), TUJ1 (1:500, Covance), MAP2 (1:400, Sigma-Aldrich),and DAPI (1:5000, Molecular Probes) and allowed to incubate for 2 hoursat room temperature. The samples were washed 3× with PBS and secondaryantibodies of Alexa-Fluor conjugated goat anti-mouse (1:200) and goatanti-rabbit (1:200) were added and allowed to incubate for a minimum of1 hour at room temperature covered with aluminum foil to protect themfrom light. The samples were washed 2× with PBS and mounted tocoverslips using Gel-mount (Invitrogen). The samples were imaged usinglaser scanning confocal microscopy.

Immunostaining results from examining the cells after 7 days in cultureon PVDF fibers stimulated with 9 mV/cm direct current applied fieldindicate advanced differentiated cell morphology by the presence ofintermediate filament vimentin. The cells attached to multiple layers ofthe scaffold with the nuclei highly aligned along the fibers. Vimentinappeared to be forming a network across the fibers similar to what wasobserved in the culture on CP2 and PVDF fibers. Neuronal markers MAP2and TUJ1 were evident.

Western Blot was performed. Determining the protein concentration wasperformed. After 7 and 14 days in culture, protein was extracted fromthe cells attached to the scaffolds by placing the dish on ice,aspirating the media, washing 1× with PBS and adding 250 μl RIPA buffer(Pierce) containing 1:100 Halt Protease Inhibitor (PI, Pierce) cocktail(100 mM AEBSF-HCl, 80 μM Aprotinin, 5 mM Bestatin, 1.5 mM E-64, 2 mMLeupeptin, 1 mM Pepstatin A) to prevent protein degradation. The lysisbuffer containing the extracted proteins was transferred to a 2 mlmicrocentrifuge tube and stored at −80° C. until use. The proteinconcentration was quantified using the bicinchoninic acid (BCA) kit(Pierce). A standard curve was generated using BSA. The Gel wasprepared, loaded and run. The protein sample containing 2× Laemmlibuffer (Sigma-Aldrich) was heated at 95° C. for 5 minutes andcentrifuged briefly prior to loading. Polyacrylamide Ready Gels (Biorad)having a range from 5-20% and 30 μl wells were placed in theelectrophoresis chamber. The chamber was filled with 1X SDS-PAGE runningbuffer consisting of Tris base (25 mM, pH 6.8), Glycine (192 mM) and SDS(0.1%) in deionized water. The standard was made from Precision PlusProtein Kaleidoscope Standard (Biorad) and 5.5 μl was loaded into thefirst well of each gel. 10 μg of protein was loaded in the gel. The gelwas run at 180 V for 55 minutes. The Gel was transferred. The gel wasremoved and placed over a pre-wetted nitrocellulose transfer membrane.The gel and transfer membrane were assembled and placed in the chamberwith the gel facing the anode and membrane facing the cathode. An iceblock was added to the chamber and it was filled with transfer bufferconsisting of Trisbase (25 mM), Glycine (192 mM) and Methanol (20%). Theset-up was transferred to the cold room and run for 90 minutes at 80 V.

Primary and Secondary Antibodies were added. A 100 ml solution of 5%nonfat milk (Biorad) in 1×TBS (50 mM Tris pH 8.0, 150 mM NaCl)containing 0.1% Tween 20 (Sigma-Aldrich) was prepared for blocking. Thetransfer membrane was removed from the chamber, stained with Ponceau Snon-specific protein stain (Sigma-Aldrich) and cut using a razor bladeat the expected molecular weight ranges for each protein being probed.The samples were placed in the milk solution to block for a minimum ofone hour. The primary antibody was added according to the followingdilutions: MAP2 (1:500), Nestin (1:500), GAPDH (1:200), Actin (1:6000),MyoD (1:200), Myogenin (1:200) and GFAP (1:500). The samples wereincubated in the primary antibody overnight in the cold room on arocker. The samples were then washed 3× with TBS containing 0.1% Tween20 for 5-10 minutes each wash. The secondary antibodies, Mouse-HRP(horseradish peroxidase) (1:5000, Novagen) and Rabbit-HRP (1:3000,Biorad), were added to a 5% milk (Biorad) solution. The samples wereincubated in the secondary antibodies for a minimum of one hour. Thesamples were then washed 3× with TBS containing 0.1% Tween 20 for 5-10minutes each wash.

Chemiluminescent Detection was performed. The antibody detectionsolution was prepared by mixing component A and B in a 40:1 ratio. Thesamples were prepared for imaging by placing Saran wrap over a piece ofcardboard and reassembling each membrane on the Saran wrap. The antibodydetection mixture was added to the membrane and allowed to incubate for5 minutes in the dark. The detection mixture was carefully blotted toremove any excess and Saran wrap was placed over the top of eachmembrane. The membrane was exposed to the camera without a filter fortwo time periods, 5 minutes and 10 minutes in the Alpha InnotechFluorochem Imager. The data was analyzed using the AlphaEase FluorChemsoftware by normalizing the protein bands to the housekeeping proteinGAPDH.

Western blot analysis showed the expression of MAP2 (72 kDa) protein instimulated PVDF fibers. Protein content was quantified by normalizing tothe housekeeping protein GAPDH. In comparison to the results obtainedfor CP2 and PVDF fibers there was much lower expression of MAP2 (72 kDa)protein for both the CP2 and PVDF fibers compared to the PVDF stimulatedfibers.

In summary, culturing hMSCs on an exemplary scaffold designed to provideelectrical stimulation succeeded in demonstrating the formation of anorganized cytoskeleton. In contrast, Titushkin et al. reported that theapplication of 2 V/cm direct current electrical stimuli to hMSCsresulted in membrane detachment from the cytoskeleton. There are severalpossible reasons for the differences observed. The field applied in ourdesign was much lower at 9 mV/cm compared to Titushkin's. Furthermore,the field applied to the PVDF fibers was used by the PVDF fibers tostimulate the dipoles in its crystalline structure resulting in anindirect effect on the cells. One could think of this as the PVDF fibersenabling the conversion of the applied voltage to a mechanical strain onthe fibers. Although this electric field was substantially lower thanwhat is required to elicit a mechanical response in the PVDF at themacroscale, any movement in the molecular structure most likely can befelt by the cells due to their highly sensitive nature.

The proliferation results indicate the stimulated PVDF fibersincorporated about 10% more EdU than the TCPS. Previous studiesinvestigating the proliferation of hMSCs when cultured on nanotopographyshow a decrease in the proliferation compared to TCPS due to theinfluence topographical cues have on the differentiation of the cells.One explanation for our results may be due to the 3D architecture of thescaffold. The cells attached to the multiple layers may have had accessto more surface area for proliferation compared to a 2D environment. Ahigh seeding density may have also caused reduced proliferation in TCPSsince hMSCs are contact inhibited cells, i.e. they stop dividing whenthey reach confluence. It would be worth repeating the experiment atdifferent seeding densities to determine the influence of thisparameter.

The results obtained for protein expression indicate the stimulated PVDFfibers are producing MAP2 protein. Immunostaining for this proteinconfirms the presence although at a low level. MAP2 has several isoformsassociated with it resulting in expression at different molecularweights, 72 kDa (MAP2c), 100 kDa (MAP2d) and kDa (MAP2a). The PVDFstimulated fibers expressed a wide band at 72 kDa but no presence of theother isoforms was detected. This may indicate the differentiationprocess is occurring at a much slower rate which correlates well withthe enhanced proliferation observed. CP2 and PVDF fibers showed strongbands for the isoform at 100 kDa and all materials showed very weak orno presence at 239 kDa. The precise role of each isoform has beendescribed by Kavallaris et al. as having distinct cellular functions.They explain that in the early stage of development and differentiation,lower molecular weight isoforms MAP2c and MAP2d form. The highermolecular weight isoforms, MAP2a and MAP2b, are found exclusively indendrites of neuronal cells with MAP2a being the highest molecularweight found in late stage development and MAP2b present in embryonicand adult stages. MAP2d is expressed as alternative splicing of MAP2, isdevelopmentally regulated and found in neuronal cell bodies. MAP2c hasbeen expressed in dendrites, axons and glial cells and has been found toexpress at high levels during early brain development. This isoform istypically replaced by higher molecular weight isoforms, however, it hasbeen found to remain in the olfactory system and the retina. Since MAP2cis found in all cell compartments, it is necessary to determine themolecular weight corresponding to the presence of MAP2 to associate thefindings with its isoform to help understand the gene expressionobserved. The presence of several isoforms for the CP2 and PVDF fiberssuggest the protein is being expressed in dendrites and the cells areassuming a neuronal lineage.

Our results have shown the application of a direct current electricfield to stimulate PVDF fibers during hMSC culture to be promising. Thedesign developed was successful though further studies are necessary todetermine the precise parameters (i.e. electric field, frequency, timeat initial voltage application, duration) to elicit any desiredeffective response. The incorporation of electrical stimuli in a 3Dscaffold by employing an electroactive polymer, such as PVDF, providesanother level of stimuli during in vitro culture and brings us closer tomimicking in vivo conditions.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of manufacturing an assembly fortissue engineering and/or stem cell differentiation comprising: (a)providing a first substrate surface comprising a first electrode; (b)electrospinning a first substantially aligned polymer fiber layer ontothe first surface; (c) independently providing one or more additionalsubstrate surfaces, each additional surface comprising an additionalelectrode; (d) independently electrospinning one or more substantiallyaligned polymer fiber layers onto each additional surface; and, (e)retaining together the layered surfaces with a clamp to provide asuspended scaffold for cell growth between the electrodes; wherein theclamped layers are retained in an orientation wherein any direct,independent electrical or mechanical stimulation to one layer will notstimulate another layer.
 2. The method of claim 1, wherein the substratesurface electrodes are prepared by fabricating a substrate from apolymer sheet and evaporating metal onto the substrate.
 3. The method ofclaim 2, wherein the polymer sheet comprises polyimide and the metalcomprises gold.
 4. The method of claim 1, wherein the electrospinningstep(s) comprise using an auxiliary electrode to create a dipole field.5. The method of claim 4, wherein the polymer fiber comprises anelectroactive polymer.
 6. The method of claim 5, wherein theelectroactive polymer comprises polyvinylidene fluoride (PVDF).